Expression,regulation, and function of alpha V integrins in hepatocellular carcinoma: an in vivo and in vitro study

The expression of alpha V integrins by neoplastic cells contributes to the promotion of local invasion and metastasis. The most characteristic extracellular ligands of alpha V integrins are vitronectin and fibronectin. Hepatocytes are the main source of vitronectin, and the capacity to synthesize and secrete vitronectin is usually retained in hepatocellular carcinoma. The aim of this study was to explore the expression, regulation, and functional role of alpha V integrins in hepatocellular carcinoma. We first analyzed the expression of alpha V integrins and their ligands fibronectin and vitronectin in 80 cases of hepatocellular carcinoma. alpha V integrin chain was detected in 44 cases and vitronectin in 50. Twenty-four of the 44 alpha V-positive tumors contained large amounts of vitronectin. These cases presented more frequently with adverse histoprognostic factors, including infiltrative growth pattern (62.5%), lack of capsule (71%), presence of capsular invasion (57%), and satellite nodules (50%). We then used HepG2 and Hep3B cell lines as in vitro models to study alpha V integrin regulation and function. HepG2 and Hep3B cells expressed alpha V integrin chain and used alpha V beta 1 and alpha V beta 5 for adhesion and migration on vitronectin. Tumor necrosis factor (TNF) alpha and transforming growth factor (TGF) beta significantly increased the expression levels of alpha V integrins and stimulated the adhesion and migration of both HepG2 and Hep3B cell lines on vitronectin. The effects of growth factors on cell adhesion and migration were reproduced by incubation with conditioned medium from rat liver myofibroblasts. In conclusion, our results support the existence of an alpha V integrin/vitronectin connection in hepatocellular carcinoma and suggest that this connection may be an adverse prognostic factor.     

Enhanced trigemino-cervical-spinal reflex recovery cycle in pain-free migraineurs

OBJECTIVE: To evaluate trigemino-cervical-spinal reflexes (TCSRs) in a group of migraine patients during the pain-free period. BACKGROUND: TCRSs are part of a complex nocifensive response involving the cervical and the upper limb muscles, and are modulated by supraspinal inhibitory pathways; it may, thus, be possible to use TCRSs to explore the trigeminal system in migraineurs. METHODS: A total of 43 migraine patients without aura (MWoA, 32 patients) or with typical aura (MWA, 11 patients) and 30 age- and sex-matched healthy subjects took part in the study. TCRSs were obtained by stimulating the supraorbital nerve and recorded from the semispinalis capitis muscle and the biceps brachii. The latency (L, msec), area (A, mVms) and recovery cycle of the reflexes were recorded. The effects of heterotopic painful stimulation on the neurophysiological parameters were studied by a validated cold pressor test (CPT). RESULTS: No significant changes were found between either migraine patients and controls or MWoA and MWA patients in the mean values in the L and A of TCRSs (t-test, P > .05). The recovery curve of the trigemino-cervical reflexes (TCRs) was significantly faster in migraine patients than in controls, while no differences were found in the trigemino-spinal reflexes (TSRs) (t-test, P < .01). Activation of the diffuse inhibitory controls through the CPT induced a significant reduction in the TCRs and TSRs area in both migraine patients and controls (paired t-test, P < .01), though the extent of this reduction did not differ significantly between migraineurs and controls (t-test, P > .05). COMMENTS: Our data suggest that the pain-free period in migraine patients is characterized by a hyperexcitability of the trigeminal pathways and of their anatomical and functional connections with the upper cervical cord neurons, and that this abnormal hyperexcitability does not appear to be due to a lack of a supraspinal inhibitory modulation.     

The Role of Rehabilitation in Patients With Progressive Supranuclear Palsy: A Narrative Review

Progressive supranuclear palsy (PSP) is a progressive neurodegenerative disorder caused by the deposition of abnormal proteins in neurons of the basal ganglia that limit motor ability, resulting in disability and reduced quality of life. So far, no pharmacologic therapy has been developed, and the treatment remains symptomatic. The aim of the present study is to perform a systematic investigation of the literature, and to determine the types and effects of rehabilitative interventions used for PSP. A search of all studies was conducted in MEDLINE/PubMed, the Cochrane Central Register of Controlled Trials, CINAHL, and EMBASE. Twelve studies were identified, including 6 case reports, 3 case series, one case-control study, one quasi?randomized trial (i.e. not truly random) with crossover design, and one randomized controlled trial, with 88 patients investigated overall. Rehabilitative interventions varied in type, number, frequency, and duration of sessions. The most commonly used clinical measures were the Progressive Supranuclear Palsy Rating Scale (PSPRS) and Unified Parkinson's Disease Rating Scale (UPDRS). Physical exercises were the main rehabilitative strategy but were associated with other interventions and rehabilitative devices, in particular treadmill and robot-assisted gait training. All studies showed an improvement in balance and gait impairment with a reduction of falls after rehabilitation treatment. Because of poor methodological quality and the variety of rehabilitative approaches including different and variable strategies, there was insufficient evidence of the effectiveness of any specific rehabilitation intervention in PSP. Despite this finding, rehabilitation might improve balance and gait, thereby reducing falls in PSP patients.

Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans

The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms.     

Regulation of bone remodeling by vasopressin explains the bone loss in hyponatremia

Although hyponatremia is known to be associated with osteoporosis and a high fracture risk, the mechanism through which bone loss ensues has remained unclear. As hyponatremic patients have elevated circulating arginine-vasopressin (AVP) levels, we examined whether AVP can affect the skeleton directly as yet another component of the pituitary-bone axis. Here, we report that the two Avp receptors, Avpr1α and Avpr2, coupled to Erk activation, are expressed in osteoblasts and osteoclasts. AVP injected into wild-type mice enhanced and reduced, respectively, the formation of bone-resorbing osteoclasts and bone-forming osteoblasts. Conversely, the exposure of osteoblast precursors to Avpr1α or Avpr2 antagonists, namely SR49059 or ADAM, increased osteoblastogenesis, as did the genetic deletion of Avpr1α. In contrast, osteoclast formation and bone resorption were both reduced in Avpr1α^−/− cultures. This process increased bone formation and reduced resorption resulted in a profound enhancement of bone mass in Avpr1α^−/− mice and in wild-type mice injected with SR49059. Collectively, the data not only establish a primary role for Avp signaling in bone mass regulation, but also call for further studies on the skeletal actions of Avpr inhibitors used commonly in hyponatremic patients.Over the past decade, studies by others and us have documented direct effects of pituitary hormones on the skeleton. We have identified functional receptors for thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), adrenocorticotropic hormone (ACTH), and oxytocin (OT) on murine and human bone cells, namely bone-forming osteoblasts and bone-resorbing osteoclasts (1–4). The genetic deletion of either the receptor or the ligand itself, as in the case of FSH and OT, results in overt skeletal abnormalities. Specifically, deleting OT or its receptor, the Oxtr, causes profound osteopenia, which primarily arises from a dramatic reduction in bone formation by the osteoblast (4). Such studies have helped establish a pituitary-bone axis, in which pituitary hormones bypass their known targets, such as the thyroid, ovaries, adrenal, and breast, to regulate bone directly (5).This growing body of data not only informs us of novel functions of pituitary hormones, but also explains the hitherto poorly understood mechanisms of certain forms of osteoporosis, which have traditionally been attributed solely to changes in distal hormones. For example, we find that low TSH signaling contributes to the bone loss in hyperthyroidism, which was thought solely to be a result of elevated thyroid hormones (6). We have also speculated that the rapid bone loss that occurs during late perimenopause, at a time when estradiol levels are relatively normal, could—at least in part—be caused by elevated serum FSH levels. Thus, an antibody to FSH reduces bone loss in ovariectomized mice by stimulating bone formation and inhibiting bone resorption (7). Similarly, through its skeletal anabolic actions, elevated OT levels during pregnancy and lactation could play a major role in enabling fetal skeletal mineralization and allowing the mother to recover from the osteoporosis caused by the intergenerational transfer of calcium (8).Here, we report studies on arginine-vasopressin (AVP), another posterior pituitary hormone, which differs from OT only by two amino acids (9). The direct skeletal actions of AVP have never been explored, despite multiple and recurring observations that hyponatremia, which is invariably accompanied by elevated plasma AVP levels, is associated with bone loss and a high fracture risk (10–16). It has been thought that, as bone is a large reservoir for sodium ions, hyponatremia will trigger sodium release from the skeleton by increasing bone resorption (17, 18). However, the molecular basis of any such effect remains unknown. Interestingly, a recent study has described a male patient with syndrome of inappropriate secretion of antidiuretic hormone- (SIADH) induced hyponatremia, who had severe osteoporosis, despite having no identifiable risk factors (19). Plasma AVP was elevated by ∼30-fold, raising the possibility that high circulating AVP levels may cause the profound bone loss.We show that AVP is a key regulator of bone resorption and formation, the two principal components of bone remodeling. Both Avp receptors, Avpr1α and Avpr2, are expressed on osteoclasts and osteoblasts, and their stimulation triggers extracellular signal regulated kinase (Erk) activation, which in turn suppresses bone formation and stimulates bone resorption. This decoupling would favor bone loss, as noted in hyponatremic states. However, we also find that the genetic deletion of Avpr1 or the pharmacologic inhibition of Avpr1 or Avpr2 increases bone mass not only by stimulating osteoblastogenesis and new bone synthesis, but also by simultaneously inhibiting osteoclast formation and bone resorption. We speculate, therefore, that the targeted therapy of hyponatremia with aquaretics (or AVPR inhibitors) could result in overall bone gain. Purposefully designed clinical studies in populations in whom hyponatremia is a significant clinical problem (20), and whom are otherwise also at a high risk for fracture (21), should shed further light on the proposed osteoprotective action of AVPR antagonists in people.     

phosphatase and tensin homolog is a differential diagnostic marker between nonalcoholic and alcoholic fatty liver disease

AIM: To investigate the protein expression of phosphatase and tensin homolog(PTEN) in human liver biopsies of patients with alcoholic and non-alcoholic liver disease.METHODS: PTEN protein expression was assessed by immunohistochemistry in formalin-fixed, paraffinembedded liver sections of patients with non-alcoholic fatty liver disease(NAFLD)(n = 44) or alcoholic liver disease(ALD)(n = 25). Liver resections obtained from 3 healthy subjects candidate for partial liver donation served as controls. Histological evaluations were performed by two experienced pathologists, and diagnoses established based on international criteria. The intensity of the PTEN staining in nuclei was compared between steatotic and non-steatotic areas of each liver fragment analyzed. For each liver specimen, the antibody-stained sections were examined and scored blindly by three independent observers, who were unaware of the patients' clinical history.RESULTS: In healthy individuals, PTEN immunostaining was intense in both the cytoplasm and nuclei of all hepatocytes. However, PTEN was strongly downregulated in both the nucleus and the cytoplasm of hepatocytes from steatotic areas in patients with NAFLD, independently of the disease stage. In contrast, no changes in PTEN protein expression were observed in patients with ALD, regardless of the presence of steatosis or the stage of the disease. The degree of PTEN downregulation in hepatocytes of patients with NAFLD correlated with the percentage of steatosis(r = 0.3061, P = 0.0459) and the BMI(r = 0.4268, P = 0.0043). Hovewer, in patients with ALD, PTEN expression was not correlated with the percentage of steatosis with or without obesity as a confounding factor(P = 0.5574). Finally, PTEN expression level in steatotic areas of ALD patients was significantly different from that seen in steatotic areas of NAFLD patients(P 0.0001).CONCLUSION: PTEN protein expression is downregulated early in NAFLD, but not in ALD. PTEN immunohistochemical detection could help in the differential diagnosis of NAFLD and ALD.     

Aldosterone and refractory hypertension: a prospective cohort study

BACKGROUND: Resistant hypertension is common in clinical practice. The aim of our study was to evaluate inappropriate aldosterone activity in causing resistance to antihypertensive therapy. METHODS: Among the patients consecutively evaluated for the first time between 1995 and 2001, we selected all those (n = 157) with an aldosterone-to-renin ratio (ARR) >or=25 (ng/dL)/(ng/mL/h), and plasma aldosterone >or=12 ng/dL. Eight patients with Conn adenoma were excluded from the study. Fifty-eight were diagnosed as idiopathic aldosteronism (IHA), the other 91 patients, who did not meet the criteria for primary aldosteronism, were operatively classified as aldosterone-associated hypertension (AAH). As a control group, we randomly chose 160 patients with essential hypertension and plasma aldosterone <12 ng/dL (EH). Antihypertensive treatment was given in accordance to World Health Organization Guidelines (1999). The study end point was blood pressure (BP) <140/90 mm Hg. RESULTS: During follow-up (22 +/- 2 months), 24 (41.4%) patients with IHA, 35 (38.5%) with AAH, and 72 (54.0%) with EH reached the end point. According to survival analysis, AAH and IHA patients reached the end point in a smaller fraction and in a longer time compared with EH patients, with no difference between IHA and AAH. At the end of follow-up, IHA and AAH patients had higher diastolic BP than EH patients with no difference between IHA and AAH. CONCLUSIONS: Patients with elevated aldosterone plasma levels develop resistant hypertension, even in the absence of clinically diagnosed primary aldosteronism. Their identification will allow a targeted therapy and a more effective BP reduction.     

Effect of painful heterotopic stimulation on the cutaneous silent period in the upper limbs.

OBJECTIVE: To evaluate the effect of heterotopic painful stimulation (HPS) on the cutaneous silent period (CSP) and the withdrawal flexor reflex (WFR) in the upper limbs, in an attempt to better identify the nociceptive component of the CSP and its functional relationship with the flexor reflex. METHODS: The CSP at different stimulus intensities, the WFR and the H/M ratio were studied in 12 healthy adults. Neurophysiological measurements were recorded in the following 4 conditions: (1) control session; (2) non-painful session (dipping hand in water at 25 degrees C); (3) painful (cold pressor test, CPT); and (4) after-effect (3-8 min after taking hand out of water at 5-6 degrees C). RESULTS: During the CPT, the duration of the high-threshold CSP was approximately 23% shorter than the baseline value, the high-threshold CSP latency was approximately 10% longer than the baseline value, and the mean RIII reflex area was approximately 40% smaller than the baseline reflex area (all P<0.05). A significant correlation was found between the percentage decrease in the CSP duration and the WFR area (r=0.61, P<0.05). CONCLUSIONS: Our findings indicate that the HPS specifically inhibits both the high-threshold CSP and the WFR, thereby providing further evidence that these cutaneous reflexes are functionally and anatomically related, and that they represent different aspects of a complex nocifensive response.